1. Field of the Invention
The present invention relates to a reagent which is effective in the determination of the presence of the chlorine ion in a substance and thus the reagent is effectively utilizable in the fields of analytical chemistry and diagnosis. More particularly, the present invention relates to a reagent for the determination of the chlorine ion, which utilizes the nature of .alpha.-amylase which is convertible from its deactivated from into its active form in the presence of the chlorine (chloride) ion.
2. Description of the Prior Art
In the field of chemistry, a great number of reports have been submitted hitherto over a century with respect to the analysis of chlorine, including a primitive routine method for the determination of the presence of the chlorine ion utilizing a soluble silver salt and the use of tolidine for the colorimetric test of free chlorine. Recently, determination of the chlorine ion is utilized in a wide variety of fields, including the precise chemical analysis and diagnosis in medical fields. Especially, determination of the chlorine ion in serum is clinically important in grasping the metabolic function of electrolytes in the living body. Methods for the determination of the chlorine ion in samples, now widely used in clinical laboratories utilize either (A) the electric methods, such as coulometric titration [velapoldi R A, Paule R C, Schaffer R et al. "A reference method for the determination of chloride in serum" Nat. Bur. Stand. (U.S.) Special Publication 260-67 (1979)] and the selective electrode method [Oesch U, Ammann D, Simon W. "Ion-selective membrane electrodes for clinical use" Clin. Chem. 1986 (32) 1448-1459], wherein the concentration of chlorine ion is measured as changes in electric signals are noted; or (B) the chemical methods, such as colorimetry [Schales O, Schales S S. "A simple and accurate method for the determination of chloride in biological fluids" J. Biol. Chem. 1941, 140, 879; Sendroy J. "Note on the Photoelectric microdetermination of chloride in biological fluids" J. Biol. Chem. 1942 142, 171-173; Zall D M, Fisher D, Garner M O. "Photometric determination of chlorides in water" Anal. Chem. 1956, (28) 1665-1668], wherein the concentration of the chlorine ion is measured according to changes in color density. Among these conventional methods for the determination of the chlorine ion, the coulometric titration method, usually employed in a manual or semi-automatic method, is considered to be the most reliable but requires time for titration. This method can hardly be set in an automatic analytical system. The ion selective electrode method involves some maintenance problem because of its silver electrodes and also fails to meet the requisites for being used in an automatic analytical system.
As a simple method for the determination of the chlorine ion, the colorimetry method is recommended since the measuring operation can be carried out with a simple device. The typical colorimetric methods quoted above are based on the principle that thiocyanate ion formed by the reaction between mercuric thiocyanate (rhodanide) and the chlorine ion forms a complex with ferric ion and gives a characteristic red orange color effective for colorimetry. The red orange color becomes deeper as the concentration of the chlorine ion becomes higher. However, particular attention has be to paid in this colorimetric method for handling chemicals for analysis because the chemicals contain the harmful mercuric ions and the thiocyanate ions. Thus, the waste solution or residue from the apparatus used in this method causes serious environmental pollution. Special devices and treatments are therefore necessary for the treatment of such waste solution and residue, thus making this colorimetric method practically less attractive.
On the other hand, it is desirable that the method for the determination of the chlorine ion can be carried out with a spectrophotometer widely employed in laboratories. Recently, it is highly desired to assemble the device for the determination of the chlorine ion in an automatic analytical system.
Under the above mentioned circumstances, there is a great demand for developing a new type method or device for the determination of the chlorine ion in a simple and precise manner using a conventional measuring device and applicable to an automatic analytical system without any problem of environmental pollution as seen in the prior art colorimetric methods.